Rate Of Decarboxylation Research Articles

Biodegradation of Cinnamates by White-Rot Fungus, Phlebia Radiata

th day of the cultivation. The evolution of carbon dioxide from aliphatic chain cleavage cinnamate showed very similar trend which demonstrated simultaneous process with the decarboxylation ones on all tested media. About 30% of carbon dioxide evolved from carboxyl-labeled cinnamate. The radioactivity in the mycelium was also above 10% in all cases and can be read as partially degraded or metabolized to the other components. The cellulose slightly repressed the decarboxylation of cinnamate in opposition to wheat straw and spruce wood, which doubled degradation. The maximum rate of decarboxylation was ca. 1.2% of the applied activity evolved as 14 CO2 per hour. According to identified metabolites, at first the substrates after decarboxylation, and aliphatic chain cleavage were demethylated in the position 4 following the demethylation in the position 3 and finally aromatic ring cleavage were observed in air and oxygen aeration on the media with glucose, cellu- lose, wheat straw and spruce wood.

Changes of Isoflavone Profile in the Hypocotyls and Cotyledons of Soybeans during Dry Heating and Germination

A gradient reversed-phase high-performance liquid chromatography (HPLC) method has been developed to be suitable for the separation and determination of 12 isoflavones in soybeans. Profiles of daidzein, genistein, glycitein, and their malonyl-, acetyl-, and nonconjugated beta-glycosides were determined in cotyledons and hypocotyls of soybeans as affected by dry heating and germination. The results showed that the compositions and concentrations of isoflavones were remarkably different in the two parts of soybeans, and hypocotyls contained a much higher content of isoflavones than cotyledons (e.g., 7.8-fold higher). In hypocotyls, daidzein and its glycoside conjugates (59.6%) were the most abundant isoflavones, being followed by glycitein (26.6%) and genistein series (13.8%). In cotyledons, genistein and its glycoside conjugates (61.9%) were the main isoflavones, being followed by daidzein series (38.1%), and no glycitein series was found. Both hypocotyls and cotyledons contained remarkably high amounts of malonylglycosides (69.1 and 69.4%, respectively) and beta-glycosides (27.1 and 25.4%), and only a very small quantity of aglycones (3.8 and 5.2%) and no acetylglycosides were detected. Acetylglycosides and beta-glycosides were the thermal decarboxylation and deesterification products, respectively, of malonylglycosides, which were thermally unstable. The relative rates of decarboxylation and deesterification reactions were different in cotyledons and hypocotyls at different temperatures. During the process of germination, beta-glycosides decreased, and malonylglycosides and aglycones increased, and then, malonylglycosides were the major fractions in germinating soybeans. Interestingly, the present study occasionally found a significant circadian change between malonylglycosides and aglycones with a nocturnal increase of aglycones and decrease of malonylglycosides during germination, and even aglycones became the most abundant forms at night. However, this mechanism is yet to be investigated.

Orotic Acid Decarboxylation in Water and Nonpolar Solvents: A Potential Role for Desolvation in the Action of OMP Decarboxylase

OMP decarboxylase (ODCase) generates a very large rate enhancement without the assistance of metals or other cofactors. The uncatalyzed decarboxylation of 1-methylorotate in water is shown to involve the monoanion, although uncharged 1-methylorotic acid is decarboxylated at a similar rate. To measure the extent to which the rate of the nonenzymatic decarboxylation of orotate derivatives might be enhanced by their removal from solvent water, the 1-phosphoribosyl moiety of OMP was replaced with 1-substituents that would allow it to enter less polar solvents. When the tetrabutylammonium salt of 1-cyclohexylorotate was transferred from water to a series of dipolar aprotic solvents, its rate of decarboxylation increased markedly, varying with the relative ability of each solvent to release the substrate in the ground state from stabilization by solvent water acting as a proton donor. These findings are consistent with the view that separation of the substrate from solvent water may contribute, at least to a limited extent, to the rate enhancement produced by ODCase. This enzyme's active site, like that of another cofactorless enzyme recently shown to produce a rate enhancement similar in magnitude (uroporphyrinogen decarboxylase), is equipped with an ammonium group positioned in such a way as to balance the electrostatic charge of the carboxylate group of the substrate and later supply a proton to the incipient carbanion in a relatively waterless environment.

Decarboxylation of fatty acids over Pd supported on mesoporous carbon

Fatty acid decarboxylation was studied in a semibatch reactor over 1 wt.% Pd/C (Sibunit) using five different fatty acids, C17–C20 and C22, as feeds. The same decarboxylation rates were obtained for pure fatty acids, whereas extensive catalyst poisoning and/or sintering and coking occurred with low purity fatty acids as reactants. One reason for catalyst poisoning using behenic acid (C22) as a feedstock was its high phosphorus content. The decarboxylation rate of fatty acids decreased also with increasing fatty acid to metal ratio.

Short‐term dynamics of isotopic composition of leaf‐respired CO2 upon darkening: measurements and implications

Recent advances in understanding the metabolic origin and the temporal dynamics in delta(13)C of dark-respired CO(2) (delta(13)C(res)) have led to an increasing awareness of the importance of plant isotopic fractionation in respiratory processes. Pronounced dynamics in delta(13)C(res) have been observed in a number of species and three main hypotheses have been proposed: first, diurnal changes in delta(13)C of respiratory substrates; second, post-photosynthetic discrimination in respiratory pathways; and third, dynamic decarboxylation of enriched carbon pools during the post-illumination respiration period. Since different functional groups exhibit distinct diurnal patterns in delta(13)C(res) (ranging from 0 to 10 per thousand diurnal increase), we explored these hypotheses for different ecotypes and environmental (i.e. growth light) conditions. Mass balance calculations revealed that the effect of respiratory substrates on diurnal changes in delta(13)C(res) was negligible in all investigated species. Further, rapid post-illumination changes in delta(13)C(res) (30 min), which increased from 2.6 per thousand to 5 per thousand over the course of the day, were examined by positional (13)C-labelling to quantify changes in pyruvate dehydrogenase (PDH) and Krebs cycle (KC) activity. We investigated the origin of these dynamics with Rayleigh mass balance calculations based on theoretical assumptions on fractionation processes. Neither the estimated changes of PDH and KC, nor decarboxylation of a malate pool entirely explained the observed pattern in delta(13)C(res). However, a Rayleigh fractionation of (12)C-discriminating enzymes and/or a rapid decline in the decarboxylation rate of an enriched substrate pool may explain the post-illumination peak in delta(13)C(res). These results are highly relevant since delta(13)C(res) is used in large-scale carbon cycle studies.

Mechanism of the Orotidine 5′-Monophosphate Decarboxylase-Catalyzed Reaction: Effect of Solvent Viscosity on Kinetic Constants

Orotidine 5'-monophosphate decarboxylase (OMPDC) is an exceptionally proficient catalyst: the rate acceleration (k(cat)/k(non)) is 7.1 x 10(16), and the proficiency [(k(cat)/K(M))/k(non)] is 4.8 x 10(22) M(-1). The structural basis for the large rate acceleration and proficiency is unknown, although the mechanism has been established to involve a stabilized carbanion intermediate. To provide reaction coordinate context for interpretation of the values of k(cat), k(cat)/K(M), and kinetic isotope effects, we investigated the effect of solvent viscosity on k(cat) and k(cat)/K(M) for the OMPDCs from Methanothermobacter thermautotrophicus (MtOMPDC) and Saccharomyces cerevisiae (ScOMPDC). For MtOMPDC, we used not only the natural OMP substrate but also a catalytically impaired mutant (D70N) and a more reactive substrate (FOMP); for ScOMPDC, we used OMP and FOMP. With MtOMPDC and OMP, k(cat) is independent of solvent viscosity, indicating that decarboxylation is fully rate-determining; k(cat)/K(M) displays a fractional dependence of solvent viscosity, suggesting that both substrate binding and decarboxylation determine this kinetic constant. For ScOMPDC with OMP, we observed that both k(cat) and k(cat)/K(M) are fractionally dependent on solvent viscosity, suggesting that the rates of substrate binding, decarboxylation, and product dissociation are similar. Consistent with these interpretations, for both enzymes with FOMP, the increases in the values of k(cat) and k(cat)/K(M) are much less than expected based on the ability of the 5-fluoro substituent to stabilize the anionic intermediate; i.e., substrate binding and product dissociation mask the kinetic effects of stabilization of the intermediate by the substituent.

Production of 1-Carbon Units from Glycine Is Extensive in Healthy Men and Women

Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield CO2, NH3, and a 1-carbon unit. CO2 also can be generated from the 2-carbon of glycine by 10-formyltetrahydrofolate-dehydrogenase and, after glycine-to-serine conversion by serine hydroxymethyltransferase, from the tricarboxylic acid cycle. To evaluate the relative fates of glycine carbons in CO2 generation in healthy volunteers (3 male, 3 female, aged 21–26 y), primed, constant infusions were conducted using 9.26 μmol·h−1·kg−1 of [1,2-13C]glycine and 1.87 μmol·h−1·kg−1 of [5,5,5-2H3]leucine, followed by an infusion protocol using [1-13C]glycine as the glycine tracer. The time period between the infusion protocols was >6 mo. In vivo rates of whole-body glycine and leucine flux were nearly identical in protocols with [1,2-13C]glycine and [5,5,5-2H3]leucine and with [1-13C]glycine and [5,5,5-2H3]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-13C]glycine tracer, breath CO2 data showed a total rate of glycine decarboxylation of 96 ± 8 μmol·h−1·kg−1, which was 22 ± 3% of whole-body glycine flux. In contrast, infusion of [1,2-13C]glycine yielded a glycine-to-CO2 flux of 146 ± 37 μmol·h−1·kg−1 (P = 0.026). By difference, this implies a rate of CO2 formation from the glycine 2-carbon of 51 ± 40 μmol·h−1·kg−1, which accounts for ∼35% of the total CO2 generated in glycine catabolism. These findings also indicate that ∼65% of the CO2 generation from glycine occurs by decarboxylation, primarily from the GCS. Further, these results suggest that the GCS is responsible for the entry of 5,10-methylenetetrahydrofolate into 1-carbon metabolism at a very high rate (∼96 μmol·h−1·kg−1), which is ∼20 times the demand for methyl groups for homocysteine remethylation.

Moderate Dietary Vitamin B-6 Restriction Raises Plasma Glycine and Cystathionine Concentrations While Minimally Affecting the Rates of Glycine Turnover and Glycine Cleavage in Healthy Men and Women

Glycine is a precursor of purines, protein, glutathione, and 1-carbon units as 5,10-methylenetetrahydrofolate. Glycine decarboxylation through the glycine cleavage system (GCS) and glycine-serine transformation by serine hydroxymethyltransferase (SHMT) require pyridoxal 5′-phosphate (PLP; active form of vitamin B-6) as a coenzyme. The intake of vitamin B-6 is frequently low in humans. Therefore, we determined the effects of vitamin B-6 restriction on whole-body glycine flux, the rate of glycine decarboxylation, glycine-to-serine conversion, use of glycine carbons in nucleoside synthesis, and other aspects of 1-carbon metabolism. We used a primed, constant infusion of [1,2-13C2]glycine and [5,5,5-2H3]leucine to quantify in vivo kinetics in healthy adults (7 males, 6 females; 20–39 y) of normal vitamin B-6 status or marginal vitamin B-6 deficiency. Vitamin B-6 restriction lowered the plasma PLP concentration from 55 ± 4 nmol/L (mean ± SEM) to 23 ± 1 nmol/L (P < 0.0001), which is consistent with marginal deficiency, whereas the plasma glycine concentration increased (P < 0.01). SHMT-mediated conversion of glycine to serine increased from 182 ± 7 to 205 ± 9 μmol·kg−1·h−1 (P < 0.05), but serine production using a GCS-derived 1-carbon unit (93 ± 9 vs. 91 ± 6 μmol·kg−1·h−1) and glycine cleavage (163 ± 11 vs. 151 ± 8 μmol·kg−1·h−1) were not changed by vitamin B-6 restriction. The GCS produced 1-carbon units at a rate (∼140–170 μmol·kg−1·h−1) that greatly exceeds the demand for remethylation and transmethylation processes (∼4–7 μmol·kg−1·h−1). We conclude that the in vivo GCS and SHMT reactions are quite resilient to the effects of marginal vitamin B-6 deficiency, presumably through a compensatory effect of increasing substrate concentration.

Effects of chronic hypoglycemia and euglycemic correction on lysine metabolism in fetal sheep

In this study, we determined rates of lysine metabolism in fetal sheep during chronic hypoglycemia and following euglycemic recovery and compared results with normal, age-matched euglycemic control fetuses to explain the adaptive response of protein metabolism to low glucose concentrations. Restriction of the maternal glucose supply to the fetus lowered the net rates of fetal (umbilical) glucose (42%) and lactate (36%) uptake, causing compensatory alterations in fetal lysine metabolism. The plasma lysine concentration was 1.9-fold greater in hypoglycemic compared with control fetuses, but the rate of fetal (umbilical) lysine uptake was not different. In the hypoglycemic fetuses, the lysine disposal rate also was higher than in control fetuses due to greater rates of lysine flux back into the placenta and into fetal tissue. The rate of CO2 excretion from lysine decarboxylation was 2.4-fold higher in hypoglycemic than control fetuses, indicating greater rates of lysine oxidative metabolism during chronic hypoglycemia. No differences were detected for rates of fetal protein accretion or synthesis between hypoglycemic and control groups, although there was a significant increase in the rate of protein breakdown (P<0.05) in the hypoglycemic fetuses, indicating small changes in each rate. This was supported by elevated muscle specific ubiquitin ligases and greater concentrations of 4E-BP1. Euglycemic recovery after chronic hypoglycemia normalized all fluxes and actually lowered the rate of lysine decarboxylation compared with control fetuses (P<0.05). These results indicate that chronic hypoglycemia increases net protein breakdown and lysine oxidative metabolism, both of which contribute to slower rates of fetal growth over time. Furthermore, euglycemic correction for 5 days returns lysine fluxes to normal and causes an overcorrection of lysine oxidation.

Hydroprocessing of Bio-Oils and Oxygenates to Hydrocarbons. Understanding the Reaction Routes

To produce diesel fuel from renewable organic material such as vegetable oils, it has for a number of years been known that triglycerides can be hydrogenated into linear alkanes in a refinery hydrotreating unit over conventional sulfided hydrodesulfurization catalysts. A number of new reactions occur in the hydrotreater, when a biological component is introduced, and experiments were conducted to obtain a more detailed understanding of these mechanisms. The reaction pathways were studied both in model compound tests and in real feed tests with mixtures of straight-run gas oil and rapeseed oil. In both sets of experiments, the hydrogenation of the oxygen containing compounds was observed to proceed either via a hydrodeoxygenation (HDO) route or via a decarboxylation route. The detailed pathway of the HDO route was further illuminated by studying the hydroprocessing of methyl laurate into n-dodecane. The observed reaction intermediates did not support a simple stepwise hydrogenation of the aldehyde formed after hydrogenation of the connecting oxygen in the ester. Instead, it is proposed that the aldehyde formed is enolized before further hydrogenation. The existence of an enol intermediate was further corroborated by the observation that a ketone lacking α-hydrogen (that cannot be directly enolized) had a much lower reactivity than a corresponding ketone with α-hydrogen. In real feed tests, the complete conversion of rapeseed oil into linear alkanes at mild hydrotreating conditions was demonstrated. From the gas and liquid yields, the relative rates of HDO and decarboxylation were calculated in good agreement with the observed distribution of the n-C17/n-C18 and n-C21/n-C22 formed. The hydrogen consumption associated with each route is deduced, and it was shown that hydrogen consumed in the water-gas-shift and methanization reactions may add significant hydrogen consumption to the decarboxylation route. The products formed exhibited high cetane values and low densities. The challenges of introducing triglycerides in conventional hydrotreating units are discussed. It is concluded that hydrotreating offers a robust and flexible process for converting a wide variety of alternative feedstocks into a green diesel fuel that is directly compatible with existing fuel infrastructure and engine technology.

Kinetic Mechanism and Structural Requirements of the Amine-Catalyzed Decarboxylation of Oxaloacetic Acid

The kinetic and chemical mechanism of amine-catalyzed decarboxylation of oxaloacetic acid at pH 8.0 has been reevaluated using a new and versatile assay. Amine-catalyzed decarboxylation of oxaloacetic acid proceeds via the formation of an imine intermediate, followed by decarboxylation of the intermediate and hydrolysis to yield pyruvate. The decrease in oxaloacetic acid was coupled to NADH formation by malate dehydrogenase, which allowed the rates of both initial carbinolamine formation (as part of the imination step) and decarboxylation to be determined. By comparing the rates observed for a variety of amines and, in particular, diamines, the structural and electronic requirements for diamine-catalyzed decarboxylation at pH 8.0 were identified. At pH 8.0, monoamines were found to be very poor catalysts, whereas some diamines, most notably ethylenediamine, were excellent catalysts. The results indicate that the second amino group of diamines enhances the rate of imine formation by acting as a proton shuttle during the carbinolamine formation step, which enables diamines to overcome high levels of solvation that would otherwise inhibit carbinolamine, and thus imine, formation. The presence of the second amino group may also enhance the rate of the carbinolamine dehydration step. In contrast to the findings of previous reports, the second amino group participates in the reaction by enhancing the rate of decarboxylation via hydrogen-bonding to the imine nitrogen to either stabilize the negative charge that develops on the imine during decarboxylation or preferentially stabilize the reactive imine over the unreactive enamine tautomer. These results provide insight into the precise catalytic mechanism of several enzymes whose reactions are known to proceed via an imine intermediate.

Uroporphyrinogen decarboxylation as a benchmark for the catalytic proficiency of enzymes

The magnitude of an enzyme's affinity for the altered substrate in the transition state exceeds its affinity for the substrate in the ground state by a factor matching the rate enhancement that the enzyme produces. Particularly remarkable are those enzymes that act as simple protein catalysts, without the assistance of metals or other cofactors. To determine the extent to which one such enzyme, human uroporphyrinogen decarboxylase, enhances the rate of substrate decarboxylation, we examined the rate of spontaneous decarboxylation of pyrrolyl-3-acetate. Extrapolation of first-order rate constants measured at elevated temperatures indicates that this reaction proceeds with a half-life of 2.3 x 10(9) years at 25 degrees C in the absence of enzyme. This enzyme shows no significant homology with orotidine 5'-monophosphate decarboxylase (ODCase), another cofactorless enzyme that catalyzes a very slow reaction. It is proposed that, in both cases, a protonated basic residue (Arg-37 in the case of human UroD; Lys-93 in the case of yeast ODCase) furnishes a counterion that helps the scissile carboxylate group of the substrate leave water and enter a relatively nonpolar environment, stabilizes the incipient carbanion generated by the departure of CO(2), and supplies the proton that takes its place.

Oxaloacetate-to-malate conversion by mineral photoelectrochemistry: implications for the viability of the reductive tricarboxylic acid cycle in prebiotic chemistry

AbstractThe carboxylic acids produced by the reductive tricarboxylic acid (rTCA) cycle are possibly a biosynthetic core of initial life, although several steps such as the reductive kinetics of oxaloacetate (OAA) to malate (MA) are problematic by conventional chemical routes. In this context, we studied the kinetics of this reaction as promoted by ZnS mineral photoelectrochemistry. The quantum efficiency φMAof MA production from the photoelectrochemical reduction of OAA followed φMA=0.13 [OAA] (2.1×10−3+[OAA])−1and was independent of temperature (5 to 50°C). To evaluate the importance of this forward rate under a prebiotic scenario, we also studied the temperature-dependent rate of the backward thermal decarboxylation of OAA to pyruvate (PA), which followed an Arrhenius behavior as log (k−2)=11.74–4956/T, wherek−2is in units of s−1. These measured rates were employed in conjunction with the indirectly estimated carboxylation rate of PA to OAA to assess the possible importance of mineral photoelectrochemistry in the conversion of OAA to MA under several scenarios of prebiotic conditions on early Earth. As an example, our analysis shows that there is 90% efficiency with a forward velocity of 3 yr/cycle for the OAA→MA step of the rTCA cycle at 280 K. Efficiency and velocity both decrease for increasing temperature. These results suggest high viability for mineral photoelectrochemistry as an enzyme-free engine to drive the rTCA cycle through the early aeons of early Earth, at least for the investigated OAA→MA step.

Global Transcription and Metabolic Flux Analysis of Escherichia coli in Glucose-Limited Fed-Batch Cultivations

A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic "work mode." sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

Allosteric Coupling in Pyruvate Dehydrogenase Kinase 2

Mitochondrial pyruvate dehydrogenase kinase 2 (PDHK2) phosphorylates the pyruvate dehydrogenase multienzyme complex (PDC) and thereby controls the rate of oxidative decarboxylation of pyruvate. The activity of PDHK2 is regulated by a variety of metabolites such as pyruvate, NAD (+), NADH, CoA, and acetyl-CoA. The inhibitory effect of pyruvate occurs through the unique binding site, which is specific for pyruvate and its synthetic analogue dichloroacetate (DCA). The effects of NAD (+), NADH, CoA, and acetyl-CoA are mediated by the binding site that recognizes the inner lipoyl-bearing domain (L2) of the dihydrolipoyl transacetylase (E2). Both allosteric sites are separated from the active site of PDHK2 by more than 20 A. Here we show that mutations of three amino acid residues located in the vicinity of the active site of PDHK2 (R250, T302, and Y320) make the kinase resistant to the inhibitory effect of DCA, thereby uncoupling the active site from the allosteric site. In addition, we provide evidence that substitutions of R250 and T302 can partially or completely uncouple the L2-binding site. Based on the available structural data, R250, T302, and Y320 stabilize the "open" and "closed" conformations of the built-in lid that controls the access of a nucleotide into the nucleotide-binding cavity. This strongly suggests that the mobility of ATP lid is central to the allosteric regulation of PDHK2 activity serving as a conformational switch required for communication between the active site and allosteric sites in the kinase molecule.

Synthesis and in situ foaming of biodegradable malonic acid ESO polymers

AbstractIn this study, biodegradable rigid cellular materials were synthesized from the reaction of malonic acid with epoxidized soybean oil. Malonic acid reacts with two epoxy groups to give a network polymer. In the course of this reaction, initially formed malonic acid monoester (MAME) can decarboxylate and produce CO2, which acts as the blowing agent leading to in situ foaming of the polymer. Epoxide addition and decarboxylation reactions of MAME occur competitively and simultaneously and by controlling their relative rates, foams of controlled density were produced. 1H NMR spectrum of the synthesized foams showed that increasing the temperature increases the rate of decarboxylation reaction of MAME and decreases crosslink density leading to softer and lower density foams. Addition of 1,4‐diazabicyclo[2.2.2]octane (DABCO) as a catalyst also increases the rate of decarboxylation. Load deflection curves of the cellular materials showed that decreasing the temperature and addition of DABCO increase compressive modulus of samples. Cell morphology was studied by microscopic images of foam samples that showed that foam samples have a closed cell structure and a wide distribution of cell volume. Soil burial test was done to determine rate of biodegradation of foam samples. A half‐life of 815 days showed that foam samples are highly biodegradable. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Tracing of glycine carbons in healthy men and women using primed, constant infusion of [1‐13C1]glycine or [1,2‐13C2]glycine and [2H3]leucine

Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield CO2, NH3, and a one‐carbon unit. CO2 also can be generated from the 2‐carbon of glycine by 10‐formyltetrahydrofolate‐dehydrogenase and, after glycine‐to‐serine conversion by serine hydroxymethyltransferase (SHMT), from the TCA cycle.To evaluate the relative fates of glycine carbons in CO2 generation in healthy volunteers (3 male, 3 female; aged 21–26y), primed, constant infusions were conducted using 9.26 μmol/(kg*h) of [1,2‐13C2]glycine and 1.87 μmol/(kg*h) of [5,5,5‐2H3]leucine, followed by an infusion protocol using [1–13C1]glycine as the glycine tracer.The rate of serine synthesis via SHMT was 186 ± 10 μmol/(kg*h) (mean ± SEM) after infusion of [1‐13C1]glycine and 185 ± 18 μmol/(kg*h) after infusion of [1,2‐13C2]glycine. Using the [1‐13C1]glycine tracer, breath CO2 data showed a glycine decarboxylation rate of 96 ± 3 μmol/(kg*h), which indicated that GCS accounted for 22 ± 1% of whole body glycine flux. Infusion of [1,2‐13C2]glycine yielded a glycine‐to‐CO2 flux of 146 ± 15 μmol/(kg*h). By difference, this implies a rate of CO2 formation from the glycine 2‐carbon of 51 ± 16 μmol/(kg*h), which accounts for ~35% of the CO2 generated in glycine catabolism. The results from the [1‐13C1]glycine tracer indicate an equally high rate (~96 μmol/(kg*h)) of production of one‐carbon units by the GCS.Supported by NIH grant DK072398 and GCRC grant M01‐RR00082

Putative occurrence of lysine decarboxylase isoforms in soybean (Glycine max) seedlings

The activity of lysine decarboxylase was studied in 3-day-old soybean (Glycine max (L.) Meer cv. Sakai) seedlings also in relation to light conditions. Lysine decarboxylase activity was mainly localized in the roots and to a lesser extent in the hypocotyls and was detectable in both the soluble and particulate fractions. The enzyme activity levels were similar during germination under light and dark conditions. With respect to lysine concentration, the initial decarboxylation rate of the soluble fraction showed a saturating curve. Conversely, the initial decarboxylation rate of the particulate fraction showed a sigmoidal curve. These results could suggest that at least two isoforms of lysine decarboxylase are present in different organs of soybean seedlings. In the root soluble fraction, the suicide inhibitor alpha-difluoromethyl-lysine suppressed the activity of lysine decarboxylase and of ornithine decarboxylase to the same extent, but had no effect on arginine decarboxylase activity.

One-Electron Oxidation of 2-(4-Methoxyphenyl)-2-Methylpropanoic and 1-(4-Methoxyphenyl)cyclopropanecarboxylic Acids in Aqueous Solution. The Involvement of Radical Cations and the Influence of Structural Effects and pH on the Side-Chain Fragmentation Reactivity

A product and time-resolved kinetic study on the one-electron oxidation of 2-(4-methoxyphenyl)-2-methylpropanoic acid (2), 1-(4-methoxyphenyl)cyclopropanecarboxylic acid (3), and of the corresponding methyl esters (substrates 4 and 5, respectively) has been carried out in aqueous solution. With 2, no direct evidence for the formation of an intermediate radical cation 2*+ but only of the decarboxylated 4-methoxycumyl radical has been obtained, indicating either that 2*+ is not formed or that its decarboxylation is too fast to allow detection under the experimental conditions employed (k > 1 x 10(7) s(-1)). With 3, oxidation leads to the formation of the corresponding radical cation 3*+ or radical zwitterion -3*+ depending on pH. At pH 1.0 and 6.7, 3*+ and -3*+ have been observed to undergo decarboxylation as the exclusive side-chain fragmentation pathway with rate constants k = 4.6 x 10(3) and 2.3 x 10(4) s(-1), respectively. With methyl esters 4 and 5, direct evidence for the formation of the corresponding radical cations 4*+ and 5*+ has been obtained. Both radical cations have been observed to display a very low reactivity and an upper limit for their decay rate constants has been determined as k < 10(3) s(-1). Comparison between the one-electron oxidation reactions of 2 and 3 shows that the replacement of the C(CH3)2 moiety with a cyclopropyl group determines a decrease in decarboxylation rate constant of more than 3 orders of magnitude. This large difference in reactivity has been qualitatively explained in terms of three main contributions: substrate oxidation potential, stability of the carbon-centered radical formed after decarboxylation, and stereoelectronic effects. In basic solution, -3*+ and 5*+ have been observed to react with -OH in a process that is assigned to the -OH-induced ring-opening of the cyclopropane ring, and the corresponding second-order rate constants (k-OH) have been obtained. With -3*+, competition between decarboxylation and -OH-induced cyclopropane ring-opening is observed at pH >or=10, with the latter process that becomes the major fragmentation pathway around pH 12.